EN
ANNEX
Part I.
Point 1. In Annex IV [to Regulation (EU) 2016/1240], point 2 is amended as follows:
In last indent "in accordance with Annex IV of Regulation 273/2008" is deleted.
Point 2. In Annex IV [to Regulation (EU) 2016/1240], the following part Ia is inserted:
'Part Ia
Methods of analysis of unsalted butter for public intervention
Parameter / MethodFat / ISO 17189
Water / ISO 3727 part 1
Solid non fat / ISO 3727part 2
Fat acidity / ISO 1740
Peroxide value / ISO 3976
Non-milk fat / ISO 17678
Sensory characteristics / ISO 22935 part 2 and 3 and scoring table hereafter.
Scoring table
Appearance / Consistency / Odour and FlavourPoints / Remarks / Points / Remarks / Points / Remarks
5 / Very good
Ideal type
Highest quality
(equal dry) / 5 / Very good
Ideal type
Highest quality
(equal spreadable) / 5 / Very good
Ideal type
Highest quality
(absolutely pure finest odour)
4 / Good
(no evident defects) / 4 / Good
(no evident defects) / 4 / Good
(no evident defects)
1, 2 or 3 / Any defect / 1, 2 or 3 / Any defect / 1, 2 or 3 / Any defect
Part II. In Annex V [to Regulation (EU) 2016/1240], the following part Ia is inserted:
'Part Ia
Methods ofanalysis of skimmed milk powder for public intervention
Parameter / MethodProtein / ISO 8968 part 1
Fat / ISO 1736
Water / ISO 5537
Acidity / ISO 6091
Lactates / ISO 8069
Phosphatase / ISO 11816 part 1
Insolubility index / ISO 8156
Scorched particles[1] / ADPI
Total bacteria count / ISO 4833-part 1
Buttermilk / Appendix I
Rennet whey / Appendix II and III
Acid whey / On the spot checks
Sensory checks [2] / ISO 22935part 2 and 3
Appendix I
SKIMMED-MILK POWDER: QUANTITATIVE DETERMINATION OF PHOSPHATIDYLSERINE AND PHOSPHATIDYLETHANOLAMINE
Method: reversed-phase HPLC
1. PURPOSE AND FIELD OF APPLICATION
The method describes a procedure for the quantitative determination of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in skimmed-milk powder (SMP) and is suitable for detecting buttermilk solids in SMP.
2. DEFINITION
PS + PE content: the mass fraction of substance determined using the procedure here specified. The result is expressed as milligrams of phosphatidylethanolamine dipalmitoyl (PEDP) per 100 g powder.
3. PRINCIPLE OF THE METHOD
Extraction of aminophospholipids by methanol from reconstituted milk powder. Determination of PS and PE as o-phthaldialdehyde (OPA) derivatives by reversed-phase (RP) HPLC and fluorescence detection. Quantification of PS and PE content in the test sample by reference to a standard sample containing a known amount of PEDP.
4. REAGENTS
All reagents must be of recognised analytical grade. Water must be distilled or water or water of at least equivalent purity, unless otherwise specified.
4.1. Standard material: PEDP, at least 99 % pure
Note: Standard material must be stored at –18 °C.
4.2. Reagents for standard sample and test sample preparation
4.2.1. HPLC-grade methanol
4.2.2. HPLC-grade chloroform
4.2.3. Tryptamine-monohydrochloride
4.3. Reagents for o-phthaldialdehyde derivatisation
4.3.1. Sodium hydroxide, 12 M water solution
4.3.2. Boric acid, 0,4 M water solution adjusted to pH 10,0 with sodium hydroxide (4.3.1)
4.3.3. 2-mercaptoethanol
4.3.4. o-phthaldialdehyde (OPA)
4.4. HPLC elution solvents
4.4.1. Elution solvents must be prepared using HPLC-grade reagents.
4.4.2. HPLC-grade water
4.4.3. Methanol of fluorimetric tested purity
4.4.4. Tetrahydrofuran
4.4.5. Sodium dihydrogen phosphate
4.4.6. Sodium acetate
4.4.7. Acetic acid.
5. APPARATUS
5.1. Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg
5.2. Beakers, 25 and 100 ml capacity
5.3. Pipettes, capable of delivering 1 and 10 ml
5.4. Magnetic stirrer
5.5. Graduated pipettes, capable of delivering 0,2, 0,5 and 5 ml
5.6. Volumetric flasks, 10, 50 and 100 ml capacity
5.7. Syringes, 20 and 100 μl capacity
5.8. Ultrasonic bath
5.9. Centrifuge, capable of operating at 27 000 × g
5.10. Glass vials, about 5 ml capacity
5.11. Graduated cylinder, 25 ml capacity
5.12. pH-meter, accurate to 0,1 pH units
5.13. HPLC equipment
5.13.1. Gradient pumping system, capable of operating at 1,0 ml/min at 200 bar
5.13.2. Autosampler with derivatisation capability
5.13.3. Column heater, capable of maintaining the column at 30 °C ± 1 °C
5.13.4. Fluorescence detector, capable of operating at 330 nm excitation wavelength and 440 nm emission wavelength
5.13.5. Integrator or data processing software capable of peak area measurement
5.13.6. A Lichrosphere — 100 column (250 × 4,6 mm) or an equivalent column packed with octadecylsilane (C 18), 5 μm particle size.
6. SAMPLING
Sampling must be carried out in accordance with ISO Standard 707.
7. PROCEDURE
7.1. Preparation of the internal standard solution
7.1.1. Weigh 30,0 ± 0,1 mg of tryptamine-monohydrochloride (4.2.3) into a 100 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1)
7.1.2. Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6) and make up to the mark with methanol (4.2.1) in order to obtain a 0,15 mM tryptamine concentration
7.2. Preparation of the test sample solution
7.2.1. Weigh 1,000 ± 0,001 g of the SMP sample into a 25 ml beaker (5.2). Add 10 ml of distilled water at 40 °C ± 1 °C by a pipette (5.3) and stir with a magnetic stirrer (5.4) for 30 minutes in order to dissolve any lumps
7.2.2. Pipette 0,2 ml (5.5) of the reconstituted milk into a 10 ml volumetric flask (5.6), add 100 μl of the 0,15 mM tryptamine solution (7.1) using a syringe (5.7) and make up to the volume with methanol (4.2.1). Mix carefully by inversion and sonicate (5.8) for 15 min
7.2.3. Centrifuge (5.9) at 27 000 g × g for 10 minutes and collect the supernatant in a glass vial (5.10)
Note: Test sample solution should be stored at 4 °C until the HPLC analysis is performed.
7.3. Preparation of the external standard solution
7.3.1. Weigh 55,4 mg PEDP (4.1) into a 50 ml volumetric flask (5.6) and add about 25 ml of chloroform (4.2.2) using a graduated cylinder (5.11). Heat the stoppered flask to 50 °C ± 1 °C and mix carefully till the PEDP dissolves. Cool the flask to 20 °C, make up to the volume with methanol (4.2.1) and mix by inversion
7.3.2. Pipette 1 ml (5.3) of this solution into a 100 ml volumetric flask (5.6) and make up to the volume with methanol (4.2.1). Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6), add 100 μl (5.7) of 0,15 mM tryptamine solution (7.1) and make up to the volume with methanol (4.2.1). Mix by inversion
Note:Reference sample solution should be stored at 4 °C until the HPLC analysis is performed.
7.4. Preparation of the derivatising reagent
Weigh 25,0 ± 0,1 mg of OPA (4.3.4) into a 10 ml volumetric flask (5.6), add 0,5 ml (5.5) of methanol (4.2.1) and mix carefully to dissolve the OPA. Make up to the mark with boric acid solution (4.3.2) and add 20 μl of 2-mercaptoethanol (4.3.3) by syringe (5.7).
Note: The derivatising reagent should be stored at 4 °C in a brown glass vial and is stable for one week.
7.5. Determination by HPLC
7.5.1. Elution solvents (4.4)
Solvent A: Solution of 0,3 mM sodium dihydrogen phosphate and 3 mM sodium acetate solution (adjusted to pH 6,5 ± 0,1 with acetic acid): methanol: tetrahydrofuran = 558:440:2 (v/v/v)
Solvent B: methanol
7.5.2. Suggested eluting gradient:
Time (min) / Solvent A (%) / Solvent B (%) / Flow rate (ml/min)Initial / 40 / 60 / 0
0,1 / 40 / 60 / 0,1
5,0 / 40 / 60 / 0,1
6,0 / 40 / 60 / 1,0
6,5 / 40 / 60 / 1,0
9,0 / 36 / 64 / 1,0
10,0 / 20 / 80 / 1,0
11,5 / 16 / 84 / 1,0
12,0 / 16 / 84 / 1,0
16,0 / 10 / 90 / 1,0
19,0 / 0 / 100 / 1,0
20,0 / 0 / 100 / 1,0
21,0 / 40 / 60 / 1,0
29,0 / 40 / 60 / 1,0
30,0 / 40 / 60 / 0
Note: The eluting gradient may require slight modification in order to achieve the resolution shown in figure 1.
Column temperature: 30 °C.
7.5.3. Injection volume: 50 μl derivatising reagent and 50 μl sample solution
7.5.4. Column equilibration
Starting up the system on a daily basis, flush the column with 100 % solvent B for 15 minutes, then set at A:B = 40:60 and equilibrate at 1 ml/min for 15 minutes. Perform a blank run by injecting methanol (4.2.1).
Note: Before long-term storage flush the column with methanol: chloroform = 80:20 (v/v) for 30 minutes.
7.5.5. Determine the PS + PE content in the test sample
7.5.6. Perform the sequence of the chromatographic analyses keeping constant the run-to-run time in order to obtain constant retention times. Inject the external standard solution (7.3) every 5-10 test sample solutions in order to calculate the response factor 0
Note: The column must be cleaned by flushing with 100 % solvent B (7.5.1) for at least 30 minutes every 20-25 runs.
7.6. Integration mode
7.6.1. PEDP peak
PEDP is eluted as a single peak. Determine the peak area by valley-to- valley integration.
7.6.2. Tryptamine peak
Tryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.
7.6.3. PS and PE peaks groups
Under the described conditions (Figure 1), PS elutes as two main partially unresolved peaks preceded by a minor peak. PE elutes as three main partially unresolved peaks. Determine the whole area of each peak cluster setting the baseline as reported in Figure 1.
8. CALCULATION AND EXPRESSION OF RESULTS
PS and PE content in the test sample shall be calculated as follows: C = 55,36 × ((A 2 )/(A 1 )) × ((T 1 )/(T 2 ))
where:
C = PS or PE content (mg/100 g powder) in the test sample
A 1 = PEDP peak area of the standard sample solution (7.3)
A 2 = PS or PE peak area of the test sample solution (7.2)
T 1 = Tryptamine peak area of the standard sample solution (7.3)
T 2 = Tryptamine peak area of the test sample solution (7.2).
9. ACCURACY OF THE METHOD
Note: The values for repeatability were calculated according to the IDF International Standard[3].
9.1. Repeatability
The relative standard derivation of the repeatability, which expresses the variability of independent analytical results obtained by the same operator using the same apparatus under the same conditions on the same test sample and in a short interval of time, should not exceed 2 % relative. If two determinations are obtained under these conditions, the relative difference between the two results should not be greater than 6 % of the arithmetic mean of the results.
9.2. Reproducibility
If two determinations are obtained by operators in different laboratories using different apparatus under different conditions for the analysis on the same test sample, the relative difference between the two results should not be greater than 11 % of the arithmetic mean of the results.
10. REFERENCES
10.1. Resmini P., Pellegrino L., Hogenboom J.A., Sadini V., Rampilli M., ‘Detection of buttermilk solids in skimmilk powder by HPLC quantification of aminophospholipids’. Sci. Tecn. Latt.-Cas., 39,395 (1988).
Figure 1
HPLC pattern of OPA-derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in methanol extract of reconstituted skim-milk powder. Integration mode for the peaks of PS, PE and tryptamine (internal standard) is reported
1
Appendix II
DETECTION OF RENNET WHEY IN SKIMMED-MILK POWDER FOR PUBLIC STORAGE BYDETERMINATION OF CASEINOMACROPEPTIDES HIGH-PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)
1. SCOPE AND FIELD OF APPLICATION
This method allows detection of rennet whey in skimmed-milk powder intended for public storage by determinationof the caseinomacropeptides.
2. REFERENCE
International Standard ISO 707 — Milk and Milk Products — Methods of sampling, conforming to the guidelinescontained in Annex I(2)I last paragraph.
3. DEFINITION
The content of rennet whey solids is defined as the percentage by mass as determined by the caseinomacropeptidecontent by the procedure described.
4. PRINCIPLE
— Reconstitution of the skimmed-milk powder, removal of fat and proteins with trichloroacetic acid, followedby centrifugation or filtration.
— Determination of the quantity of caseinomacropeptides (CMP) in the supernatant by high-performance liquidchromatography (HPLC).
— Evaluation of the result obtained for the samples by reference to standard samples consisting of skimmedmilkpowder with or without the addition of a known percentage of whey powder.
5. REAGENTS
All reagents must be of recognised analytical grade. The water used must be distilled water or water of at leastequivalent purity.
5.1. Trichloroacetic acid solution
Dissolve 240 g of trichloroacetic acid (CCl3COOH) in water and make up to 1 000 ml. The solution should beclear and colourless.
5.2. Eluent solution, pH 6,0
Dissolve 1,74 g of dipotassium hydrogen phosphate (K2HPO4), 12,37 g of potassium dihydrogen phosphate(KH2PO4) and 21,41 g of sodium sulphate (Na2SO4) in about 700 ml of water. Adjust, if necessary, to pH 6,0,using a solution of phosphoric acid or potassium hydroxide.
Make up to 1 000 ml with water and homogenise.
Note: The composition of the eluent can be updated to comply with the certificate of the standards or the recommendationsof the manufacturer of the column packing material.Filter the eluent solution, prior to use, through a membrane filter with a 0,45 μm pore diameter.
5.3Flushing solvent
Mix one volume acetonitrile (CH3CN) with nine volumes water. Filter the mixture prior to use through a membranefilter with a 0,45 μm pore diameter.
Note: Any other flushing solvent with a bactericidal effect which does not impair the columns’ resolution efficiencymay be used.
5.4. Standard samples
5.4.1. Skimmed-milk powder meeting the requirements of this Resolution (i.e. [0])
5.4.2. The same skimmed-milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard composition (i.e. [5]).
6. APPARATUS
6.1. Analytical balance
6.2. Optional centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered or capped centrifugetubes of about 50 ml capacity
6.3. Mechanical shaker
6.4. Magnetic stirrer
6.5. Glass funnels, diameter about 7 cm
6.6. Filter papers, medium filtration, diameter about 12,5 cm
6.7. Glass filtration equipment with 0,45 μm pore diameter membrane filter
6.8. Graduated pipettes allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835) or a dispensing system capable ofdelivering 10,0 ml in two minutes
6.9. Dispensing system capable of delivering 20,0 ml water at ca. 50 °C
6.10. Thermostatic water bath, set at 25 ± 0,5 °C
6.11. HPLC equipment, consisting of:
6.11.1. Pump
6.11.2. Injector, hand or automatic, with a 15 to 30 μl capacity
6.11.3. Two TSK 2 000-SW columns in series (length 30 cm, internal diameter 0,75 cm) or equivalent columns (e.g.single TSK 2 000-SWxl, single Agilent Technologies Zorbax GF 250) and a precolumn (3 cm × 0,3 cm) packedwith I 125 or material of equivalent effectiveness
6.11.4. Thermostatic column oven, set at 35 ± 1 °C
6.11.5. Variable wavelength UV detector, permitting measurements at 205 nm with a sensitivity of 0,008 A
6.11.6. Integrator capable of valley-to-valley integration
Note: Working with columns kept at room temperature is possible, but their power of resolution is slightly lower.In that case, the temperature should vary by less than ± 5 °C in any one range of analyses.
7. SAMPLING
7.1. Samples must be taken in accordance with the procedure laid down in International Standard ISO 707. However,Member States may use another method of sampling provided that it complies with the principles of the abovementionedstandard.
7.2. Store the sample in conditions which preclude any deterioration or change in composition.
8. PROCEDURE
8 . 1 . Preparation of the test sample
Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted with anairtight lid. Close the container immediately. Mix the milk powder well by means of repeated inversion of thecontainer.
8.2. Test portion
Weight 2,000 ± 0,001 g of test sample into a centrifuge tube (6.2) or a suitable stoppered flask (50 ml).
8.3. Removal of fat and proteins
8.3.1. Add 20,0 ml of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using amechanical shaker (6.3). Place the tube into the water bath (6.10) and allow to equilibrate to 25 °C
8.3.2. Add 10,0 ml of the trichloroacetic acid solution (5.1) of ca. 25 °C in two minutes, while stirring vigorously withthe aid of the magnetic stirrer (6.4). Place the tube in a water bath (6.10) and leave for 60 minutes
8.3.3. Centrifuge (6.2) for 10 minutes at 2 200 g, or filter through paper (6.6), discarding the first 5 ml of filtrate
8.4. Chromatographic determination
8.4.1. Inject 15 to 30 μl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus (6.11) operatingat a flow rate of 1,0 ml of eluent solution (5.2) per minute
Note 1. Another flow rate may be used, dependent of the internal diameter of the columns used or the instructionsof the manufacturer of the column.
Note 2. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2).
Prior to any interruption of more than 24 hours, rinse the columns with water then wash them with solution(5.3) for at least three hours at a flow rate of 0,2 ml per minute.
8.4.2. The results of chromatographic analysis of the test sample [E] are obtained in the form of chromatogram in whicheach peak is identified by its retention time RT as follows:
The choice of the column(s) can effect the retention times of the individual peaks considerably.
The integrator (6.11.6) automatically calculates the area A of each peak:
It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order todetect any abnormalities due either to malfunctioning of the apparatus or the columns, or to the origin and natureof the sample analysed.
If in doubt, repeat the analysis.
Calibration
8.5.1. Apply exactly the procedure described from point 8.2 to point 8.4.2 to the standard samples (5.4)
Use freshly prepared solutions, because CMP degrade in an 8 % trichloroacetic environment. The loss is estimatedat 0,2 % per hour at 30 °C.
8.5.2. Prior to chromatographic determination of the samples, condition the columns by repeatedly injecting the standardsample (5.4.2) in solution (8.5.1) until the area and retention time of the peak corresponding to the CMP areconstant
8.5.3. Determine the response factors R by injecting the same volume of filtrates (8.5.1) as used for the samples
9. EXPRESSION OF RESULTS
9.1. Method of calculation and formulae
9.1.1. Calculation of the response factors R:
where:
RII = the response factors of peaks II,
AII [0] = the areas of peaks II of the standard sample [0] obtained in 8.5.3.
where:
RIII = the response factor of peak III,
AIII [0] and AIII [5] = the areas of peak III in standard samples [0] and [5] respectively obtained in 8.5.3,
W = the quantity of whey in standard sample [5], i.e. 5.
9.1.2. Calculation of the relative area of the peaks in the sample [E]
SII[E] = RII × AII[E]
SIII[E] = RIII × AIII[E]
SIV[E] = RIV × AIV[E]
where:
SII [E], SIII [E], SIV [E] = the relative areas of peaks II, III and IV respectively in the sample [E],
AII [E], AIII [E] = the areas of peaks II and III respectively in the sample [E] obtained in 8.4.2,
RII, RIII = the response factors calculated in 9.1.1.
9.1.3. Calculation of the relative retention time of peak III in sample [E]: RRTIII[E] = (RTIII[E])/(RTIII[5])
where:
RRTIII [E] = the relative retention time of peak III in sample [E],
RTIII [E] = the retention time of peak III in sample [E] obtained in 8.4.2,
RTIII [5] = the retention time of peak III in control sample [5] obtained in 8.5.3.
9.1.4. Experiments have shown that there is a linear relation between the relative retention time of peak III, i.e. RRTIII [E]
and the percentage of whey powder added up to 10 %
— The RRTIII [E] is < 1,000 when the whey content is > 5 %;
— the RRTIII [E] is ≥ 1,000 when the whey content is ≤ 5 %.
The uncertainty allowed for the values of RRTIII is ± 0,002.
Normally the value of RRTIII [0] deviates little from 1,034. Depending on the condition of the columns, the value
may approach 1,000, but it must always be greater.
9.2. Calculation of the percentage of rennet whey powder in the sample:
W = SIII[E] − [1, 3 + (SIII[0] − 0, 9)]
where:
W = the percentage m/m of rennet whey in the sample [E];
SIII [E] = the relative area of peak III of test sample [E] obtained as in 9.1.2;
1,3 = represents the relative average area of peak III expressed in grams of rennet whey per
100 g determined in non-adulterated skimmed-milk powder of various origins. This figure
was obtained experimentally;
SIII [0] = represents the relative area of peak III which is equal to RIII × AIII [0]. These values areobtained in 9.1.1 and 8.5.3 respectively;
(SIII [0] − 0,9) = represents the correction to be made to the relative average area 1,3 when SIII [0] is not
equal to 0,9. Experimentally the relative average area of peak III of the control sample [0]
is 0,9.
9.3. Accuracy of the procedure
9.3.1. Repeatability
The difference between the results of two determinations carried out simultaneously or in rapid succession by thesame analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.
9.3.2. Reproducibility
The difference between two single and independent results, obtained in two different laboratories on identical test
material shall not exceed 0,4 % m/m.
9.4. Interpretation
9.4.1. Assume the absence of whey if the relative area of peak III, SIII [E] expressed in grams of rennet whey per 100 g ofthe product is ≤ 2,0 + (SIII[0] − 0,9) where
2,0 = is the maximum value allowed for the relative area of peak III taking into account the relative
area of peak III, i.e. 1,3, the uncertainty due to variations in the composition ofskimmed-milk powder and the reproducibility of the method (9.3.2),
(SIII [0] − 0,9) = is the correction to be made when the area SIII [0] is different from 0,9 (see point 9.2)
9.4.2. If the relative area of peak III, SIII [E] is > 2,0 + (SIII[0] − 0,9) and the relative area of peak II, SII [E] ≤ 160, determine